ATP sensitive potassium channels (KATP) in surface membrane (sKATP) and mitochondria (mKATP) of heart have both been implicated in the protective phenomenon called ischemic preconditioning (IPC). Proposed mechanisms for IPC are complex. The relative importance of mKATP and sKATP for IPC is controversial, in part because of heavy reliance on KATP pharmacology as well as the unknown molecular nature of mKATP. The sulfonylurea receptor subunit SUR2 regulates pharmacology and nucleotide interactions for sKATP, but the role of SUR2 in mKATP is unknown. In the last project period our goal was to develop transgenic mouse models that ablated SUR2 to determine the role of SUR2 in IPC and mKATP. Using three independent models of IPC, we found that an SUR2 mutant mouse was constitutively protected from ischemia even in the absence of IPC. This was surprising in view of the usual finding that KATP opening by drugs induced protection and that KATP block by drugs abrogated protection. We used a newly created panel of SUR2 antibodies and discovered novel short variants (Kd120, 68, 55, 28) in mouse heart mitochondria, with the SUR2-55 form uniquely in mitochondria and not the surface. The mutant surprisingly retained SUR2-68 and 55, but not 120 and 28; it was only a partial knockout. We determined the sequence of SUR2-55 and showed it persisted in the mutant because of a novel intraexonic splicing event that jumped the disruption vector. We also found that IPC in WT mice down-regulated SUR2 except for SUR2-68 and SUR2-55A, a pattern similar to the mutant mouse. We hypothesize that mKATP subunits SUR2-68 and 55A when unopposed by other SUR2 subunits confer ischemic protection. The results also make the provocative suggestion that pharmacology of mKATP may change during IPC. The role of these novel mitochondrial variants of SUR2 in the mechanism of IPC, mKATP, and mitochondrial function will be investigated in five aims. Aim 1 will determine the mechanisms for targeting of SUR2-variants to mitochondria, and mechanisms for persistence or down-regulation in IPC. Aim 2 will determine the associations of SUR2 variants with other proteins in the mKATP multi-protein complex. Aim 3 will assess the pharmacology and physiology of SUR2 variants combined with pore forming subunits in COS- 1 cells to allow interpretation of mechanisms suggested by the pharmacology of IPC. Aim 4 will investigate IPC in transgenic mice designed to have full and selective ablation of cardiac SUR2 and create specific rescue of mSUR2 variants to define mechanisms of protection. Aim 5 will determine mKATP and mitochondrial function in these mice to determine mechanisms of mSUR2 effects on mKATP, mitochondrial function, and IPC. The molecular nature of mKATP is a key gap in knowledge, and its role in IPC is controversial. This proposal utilizes transgenic animals as a novel non-pharmacological approach to fill these gaps and address the controversy. The results may suggest more specific therapeutic targets in the SUR2 variants, and it will also provide important new information on mitochondrial structure and function in heart.